Amount of ascites impacts survival in patients with hepatocellular carcinoma undergoing transarterial chemoembolization advocating for volumetric assessment

Preliminary work has shown that portal hypertension plays a key role for the prognosis in patients with hepatocellular carcinoma (HCC) undergoing transarterial chemoembolization (TACE). Specifically, the presence of ascites appears to be a strong negative predictor for these patients. However, it remains unclear whether different ascites volumes influence prognosis. Therefore, the aim of this work was to investigate the influence of different ascites volumes on survival for patients with HCC undergoing TACE. A total of 327 treatment-naïve patients with HCC undergoing initial TACE at our tertiary care center between 2010 and 2020 were included. In patients with ascites, the fluid was segmented, and the volume quantified by slice-wise addition using contrast-enhanced CT imaging. Median overall survival (OS) was calculated and univariate and multivariate Cox regression analysis has been performed. Ascites was present in 102 (31.9%) patients. Ascites volume as continuous variable was significantly associated with an increased hazard ratio in univariate analysis (p < 0.001) and remained an independent predictor of impaired median OS in multivariate analysis (p < 0.001). Median OS without ascites was 17.1 months, and therefore significantly longer than in patients with ascites (6.4 months, p < 0.001). When subdivided into groups of low and high ascites volume in relation to the median ascites volume, patients with low ascites volume had a significantly longer median OS (8.6 vs 3.6 months, p < 0.001). Ascites in patients with HCC undergoing TACE is strongly associated with a poor prognosis. Our results show that not only the presence but also the amount of ascites is highly relevant. Therefore, true ascites volume as opportunistic quantitative biomarker is likely to impact clinical decision-making once automated solutions become available.


INR
International normalized ratio IQR Interquartile range OS Overall survival TACE Transarterial chemoembolization Hepatocellular carcinoma (HCC) is among the most common cancer entities, and it ranks second among diseases responsible for cancer-related deaths 1,2 .The European Association for the Study of the Liver (EASL) and the American Association for the Study of Liver Diseases (AASLD) guidelines have recommended the Barcelona Clinic Liver Cancer (BCLC) classification system as framework for patient stratification, treatment allocation, and prognosis prediction 3,4 .According to the BCLC classification, transarterial chemoembolization (TACE) is recommended for patients with intermediate stage HCC 5 .Additionally, in the concept of stage migration and individual treatment decision-making, TACE is also applied in other BCLC stages and ranks among the most commonly applied treatments for HCC worldwide 6 .
A large amount of patients develop HCC as a consequence of liver cirrhosis 3 .Liver cirrhosis causes progressive changes in the splanchnic circulation, which lead to an increase in portal pressure 7 .In addition to portal hypertension, many patients with liver cirrhosis suffer from a retention of sodium and water 8 .In combination, both factors lead to the development of ascites, which has a negative impact on the prognosis of these patients 8 .Factors associated with portal hypertension have also been identified as highly relevant to prognosis in patients with HCC 9 .In particular, the presence of ascites has a major influence on the prognosis of patients with HCC undergoing TACE 10 .However, this does not take into account the different amounts of ascites: While some patients only have a narrow seam of ascites around the liver or spleen, other patients have large amounts of ascites that fill the entire abdominal cavity.
To date, it is unclear what role the amount of ascites plays in the prognosis of patients.Our hypothesis was that besides the presence of ascites itself, the amount of ascites also plays a critical prognostic role.Therefore, the aim of this study was to investigate the prognostic role of precise ascites volume in patients with HCC undergoing TACE (Fig. 1).

Quantified ascites volume
Among patients with ascites, the median volume was 613.5 ml (IQR 111.8-1559.8 ml).The distribution of the ascites volume is shown in Fig. 2.Among the 102 patients with ascites, 39 (38.2%) patients were within BCLC stage C or D. Of those, nine patients had at least one extrahepatic metastasis.One of these patients had peritoneal metastases.
The ascites volume showed a significant correlation with albumin (moderate correlation) and bilirubin (weak correlation), while there was no significant correlation with the thrombocyte count, INR, the tumor diameter and the number of tumors (Table 2).

Survival analysis
Figure 3 illustrates the significant correlation (p < 0.001) of ascites volume and overall survival for the individual patients.
A univariate Cox hazard regression analyses identified ascites volume, apart from AFP, albumin, bilirubin, AST, tumor count, and tumor size, as significant prognostic factor (Table 3).None of the other included risk factors showed a significant association with OS.In the subsequent multivariate analysis, ascites volume remained an independent prognostic factor, as well as albumin, bilirubin, and AST.
When stratifying patients according to the presence of ascites, patients with ascites had a significantly impaired median OS compared to patients without ascites (6.4 vs 17.1 months, p < 0.001, Fig. 4A).In the next step, the patients were divided into a group with a low and a high ascites volume depending on the median (cut off 613.5 ml ascites).Patients with a high ascites volume had a significantly impaired survival (3.6 months) compared to patients with low ascites volume (8.6 months, p < 0.001, Fig. 4B).Patients with low ascites volume had a significantly impaired survival compared to patients with no ascites (p = 0.002, Fig. 4B).
When using the cut-off value of 300 ml previously suggested for patients with liver cirrhosis, patients with ascites above this value still had a significantly impaired survival compared to patients with ascites below this cut-off (4.0 vs 9.9 months, p < 0.001).Further, patients with ascites below a volume of 300 ml had a significantly impaired survival compared to patients without ascites (17.1 months, p = 0.02).
Subsequently, subgroup analysis was performed among 166 (50.8%) patients within BCLC stage B (i.e., the recommended TACE subgroup) yielding the same results 3,4 : Patients with a high ascites volume had a significantly impaired survival (5.0 months) compared to patients with low ascites volume (12.3 months, p < 0.001, Fig. 5).Patients with low ascites volume had a significant impaired survival compared to patients with no ascites (20.1 months, p = 0.04, Fig. 5).

Risk of hepatic decompensation
We further assessed the risk of an increase in the ALBI grade for patients who had an initial grade of 1 or 2 and an available follow-up ALBI value three months after TACE (n = 197/327 (60.2%)).Of these patients, a total of n = 61 (31.0%) patients had an ALBI grade increase three months after TACE.The relative number of patients who showed an increase in the ALBI grade was significantly higher in patients with ascites than without (n = 23/37 (62.2%) vs n = 38/160 (23.8%), p < 0.001).Notably, the majority of patients with ascites already had an initial ALBI grade of 3 (n = 57/102, 56%), therefore no further increase was possible.In the remaining subset of patients with ascites and an ALBI grade of 2, numbers were small with 23 patients showing an increase from grade 2-3 and 14 patients remaining at grade 2; in these small subsets the volume distributions did not differ significantly (p = 0.26).

Discussion
In this study, we assessed the prognostic role of volumetric ascites quantification in patients with HCC undergoing TACE.We could show that the volume of ascites was associated both with poor survival outcome and with the likelihood to receive multiple TACE sessions.Table 1.Baseline patient characteristics.a More than one etiology was possible for liver disease; thus, percentages were not calculated.b In the subset of patients with ascites (n = 225).Abbreviations: NASH, nonalcoholic steatohepatitis; AIH, autoimmune hepatitis; PBC, primary biliary cholangitis; PSC, primary sclerosing cholangitis; BCLC, Barcelona Clinic Liver Cancer; AST, aspartate aminotransferase; ALT, alanine aminotransferase; AFP, alpha fetoprotein.c Sorafenib (n = 33), lenvatinib (n = 13), selective internal radiation therapy (n = 12), atezolizumab in combination with bevacizumab (n = 6), pembrolizumab (n = 2), pembrolizumab in combination with regorafenib (n = 2), lenvatinib followed by sorafenib (n = 1), linifanib followed by sorafenib (n = 1), nivolumab (n = 1), ramucirumab (n = 1).www.nature.com/scientificreports/While presence of ascites has been shown to be a prognostic factor for patients with HCC undergoing TACE treatment, combined therapy of TACE and apatinib or transarterial radioembolization, the literature regarding the influence of ascites quantification on OS in patients with HCC undergoing TACE is scarce [11][12][13] .For patients with cirrhosis, stratification into groups without ascites, ≤ 300 ml, and > 300 ml has been previously proposed 14 .
While our results also show that categorization into these volume categories result in a significant stratification regarding OS, in hazard regression analysis, the amount of ascites carried a significantly increased hazard ratio, underlining the importance of obtaining the true volume of ascites.Moreover, the volume of ascites was a significant predictor of OS in multivariate analysis when accounting for established tumor-related and liver function-related risk factors.
Until today, ascites is rarely quantified in clinical routine, due to the time-consuming nature of manual segmentations.Therefore, most scores rely on qualitative assessments, as employed in the Child-Pugh Score in sonography 15 .In recent years, several semi-quantitative scores have been developed as well, counting for example the number of abdominal regions where free fluid can be detected 16 .
Nowadays, organ segmentation and volume assessments can be performed by deep-learning algorithms.AIbased tools have the advantage that automated quantitative reports can be easily integrated into clinical workflows and can be automatically sent to the local hospital information system or image archiving and communication system 17 .If commercial products are not yet available or their implementation is not feasible, there is an initial threshold to train and validate custom solutions, in terms of labeling the appropriate datasets, computation power, and software tools.However, the latter is reduced thanks to publicly available software packages and libraries 18 .
In our cohort, free fluid was manually segmented on axial CT imaging.While this approach itself is not feasible in clinical routine, the data obtained this way forms the basis of a labeled training dataset, which will  be used to train a deep-learning algorithm, more specifically a 3D-UNet, for automated ascites detection and segmentation at our institution, as has been done for other segmentation tasks including splenic volume assessment 17 .Once trained, accurate ascites quantification will become available for other patient populations without further manual input.
In our study, ascites volume prior to TACE was lower in patients who were able to receive multiple TACE sessions.Therefore, ascites volume might serve as a quantifiable marker to assess TACE suitability in patients with unresectable HCC.With the advent of immunotherapy, the optimal time-point for a treatment switch in the concept of stage migration can be hard to identify; however, it is worth assessing as "an inappropriately high number of TACE sessions delays the switch to systemic therapy and may, in some cases, completely hinder the treatment switch due to the deterioration of liver function" 19,20 .
Moreover, development of early ascites after TACE has been shown to be associated with impaired prognosis 21 .This is in line with prior findings that have linked repeated TACE to an increase in portal hypertension and that portal hypertension is a predictor for adverse outcome in patients with liver cirrhosis and HCC [22][23][24] .
Thus, we believe that volumetric ascites quantification as opposed to visual or semi-quantitative assessments will become part of the routine work-up for cirrhotic patients and patients with liver tumors in general and for patients with HCC in particular, given the concomitant occurrence of an aggressive tumor entity and  www.nature.com/scientificreports/an underlying liver disease.In our cohort, true ascites volume was an additional parameter to identify patients with poor survival and those likely to receive multiple TACE treatments.However, future largescale multicentric evaluations are needed to determine standard reference or cut-off values for this currently underused parameter.The present study had several limitations.First, it was performed at a single-center and in a retrospective manner.However, the sample size was comparative to previous studies on this topic 14 .Additionally, our dataset was well investigated and we only included patients with complete clinical, laboratory, and imaging data.To www.nature.com/scientificreports/avoid a time bias, only patients from 2010 and later were included.These criteria minimized differences in the diagnosis and treatment decisions, which provided a more homogeneous study cohort.Moreover, similar to the BCLC classification in which the Child-Pugh Score is incorporated at initial diagnosis to give recommendations for treatment and survival estimates, ascites was volumetrically assessed in our study at initial diagnosis as well.Therefore, management of ascites in the course of disease like medication or paracentesis was not taken into account.
Another limitation is that ascites due to portal hypertension and/or peritoneal carcinomatosis may have different prognostic relevance.However, since only one patient in our study had peritoneal metastases, this could not be further investigated in this study.
Furthermore, patients that underwent previous treatments were excluded.Second, we included patients that underwent either conventional or drug-eluting bead-delivered TACE.However, several previous studies have shown OS was not influenced by the TACE delivery technique [25][26][27] .
Thus, we concluded that ascites in patients with HCC undergoing TACE is associated with a poor prognosis.Our results show that not only the presence but also the amount of ascites is highly relevant.Therefore, once automated software solutions are available, true ascites volume is likely to impact clinical decision making and to overcome its status of currently underappreciated imaging biomarker.

Methods
The Ethics Committee of the Medical Association of Rhineland Palatinate, Mainz, Germany approved this study and waived requirement for informed consent for the retrospective analysis of clinical data (permit number 2021-15,984).This study was conducted following the ethical principles of the Declaration of Helsinki.Patient records and information were anonymized prior to analysis.

Patients
A total of 714 patients with confirmed HCC that received TACE treatment in our tertiary care center between January 2010 and November 2020 were identified.Of these, 327 patients fulfilled the following inclusion criteria: (1) age above 18 years; (2) histologically or image-derived HCC diagnosis based on the EASL criteria; (3) no treatment performed prior to TACE; (4) no liver transplantation or tumor resection during the follow-up period after TACE; (5) pre-interventional contrast-enhanced CT scan for ascites volume assessment; (6) full availability of clinical, laboratory, and imaging data.A total of 387 patients were excluded, due to reasons shown in Fig. 1, and 327 treatment-naïve patients finally included.

Diagnosis, treatment, and follow-up
HCC was diagnosed based on histological or image-derived criteria established by the EASL 3,28 .All patients underwent contrast-enhanced CT for treatment planning.Indications for TACE were discussed in an interdisciplinary tumor board, which included hepatologists/oncologists, diagnostic and interventional radiologists, visceral surgeons, pathologists, and radiation therapists.TACE was performed in a standardized manner as

Figure 1 .
Figure 1.Flowchart of the patient selection process for this study.

Figure 2 .
Figure 2. Distribution of the ascites volumes among all patients with ascites (n = 102).

Figure 3 .
Figure 3. Correlation of ascites volume and overall survival.

Figure 4 .
Figure 4. (A) Kaplan-Meier curves showing survival of patients with ascites and without ascites; (B) Kaplan-Meier curves showing survival of patients with no ascites, low ascites volume, and high ascites volume in all patients (n = 327).

Figure 5 .
Figure 5. Kaplan-Meier survival curves show survival of patients with no ascites, low ascites volume and high ascites volume in the subgroup of patients within BCLC stage B (n = 166).
ParameterCorrelation coefficient p value

Table 3 .
Univariate and multivariate Cox regression results of factors related to survival for all patients (n = 327).Significant values are in bold.